ES2389751T3 - A new biomarker to monitor disease development and evaluate the efficacy of therapies - Google Patents

Abstract

A method to monitor the development of a disease in a patient, wherein said disease is selected from the group comprising: a) tissue trauma b) a reperfusion injury as a result of myocardial infarction or stroke, organ transplantation or any other surgical operation, c) cancer or cancer metastasis, and d) an inflammatory state, and where the CD73 extracted from a tissue fluid of said patient is used as a biomarker, where the method is repeated in two or more times and the CD73 level of the sample, with that of a previous analysis, and is used to indicate the worsening of the disease or to indicate the remission of the disease, where an increase in the level of the CD73 indicates remission of the disease and a decrease in the level of the CD73 indicates worsening of the illness.

Description

A new biomarker to monitor the development of diseases and evaluate the effectiveness of therapies.

FIELD OF THE INVENTION

This invention relates to the use of the CD73 protein in tissue fluids as a biomarker to assess disease development. Methods for determining CD73 protein in a tissue fluid are also described.

BACKGROUND OF THE INVENTION

Publications and other materials used herein to illustrate the basis of the invention, and in particular, cases to provide additional details regarding the practice of the invention are incorporated by reference.

CD73 is a cell surface enzyme that has 5'-ectonucleotidase activity. Thus, the conversion of monophosphorylated purine nucleotides into the corresponding nucleosides. For example, the dephosphorylation of AMP to adenosine is catalyzed by CD73. CD73 is also present in a soluble form in plasma and the soluble enzyme has the same enzymatic activity as the membrane bound form.

Adenosine is one of the physiological regulators of endothelial cell permeability [1,2], and therefore may be involved in the pathogenesis of many disorders such as acute lung injury (APL), systemic inflammatory response syndrome (SRIS), respiratory distress (RDS) and altitude sickness. In inflammation, trauma and cancer, changes in endothelial permeability also occur. CD73 controls endothelial permeability via an adenosine-mediated mechanism under normal conditions, in hypoxia and in lung injury induced by a breathing apparatus [3-7].

CD73 is induced by certain cytokines. Most importantly, it has been described that alpha and beta interferons increase the expression and activity of CD73 in humans (8, WO 2004/084933, 11). These cytokines are also used in clinics to treat different diseases. Interferon alfa, for example, is used to treat certain infections and malignant diseases, such as hepatitis and hair cell leukemia. Interferon beta, on the other hand, is widely used to treat inflammation in multiple sclerosis. However, in many cases the beneficial response to interferon treatment can only be seen in a subpopulation of patients, and also patients who respond initially may later be refractory to treatment. Therefore, there is a need to develop easily measurable biomarkers that reflect the body's biological responsiveness to treatment.

Published patent application WO 2004/084933 describes the use of cytokines to induce endothelial expression of CD73 and subsequently raise the level of adenosine in an individual. The use of interferon beta in combination with adenosine monophosphate (AMP) in the treatment of multiorganic insufficiency (IMO) in rats is described.

Published patent application WO 2007/042602 describes the use of pure beta interferon for the treatment or prevention of ischemia-reperfusion injury (LIR) or multiorganic insufficiency (IMO).

It is known that statins, hypolipidemic agents used to lower cholesterol levels induce the expression of CD73 in patients.

The use of CD73 as a marker of both general and local inflammation in rheumatoid arthritis [12] is also known.

However, in the prior art there are no descriptions concerning the measurement of serum CD73 protein or any other tissue fluid for use as a biomarker to monitor disease development or to evaluate the efficacy of therapies.

SUMMARY OF THE INVENTION

The authors of the present invention have demonstrated that the activity of soluble CD73 can be used to monitor the severity and responsiveness of a disease to therapy. Therefore, we believe that the analysis of the level of expression or activity of CD73 by any technique can provide valuable information on the course of a disease or the response to treatment.

The present invention relates to a method for monitoring the development of a disease in a patient, wherein said disease is selected from the group consisting of:

a) tissue trauma

b) reperfusion injury resulting from myocardial infarction or stroke, organ transplantation or any other

surgery,

c) cancer or cancer metastasis, and

d) an inflammatory state,

and where the CD73 extracted from a tissue fluid of said patient is used as a biomarker, wherein the method is repeated in two or more times and the level of CD73 of the sample is compared, with that of a previous analysis, and used to indicate the worsening of the disease or to indicate the remission of the disease, where an increase in the level of the CD73 indicates remission of the disease and a decrease in the level of the CD73 indicates worsening of the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Ischemia reperfusion injury of the intestine induces primary and secondary tissue damage. The mesenteric artery was occluded for 30 minutes and then reperfusion (restoration of blood flow) was allowed for 4 hours. Representative photomicrographs of the (A) intestine and (B) lungs of wild-type mice (TN, that is, not genetically modified) after false operation (laparotomy only) and after induction of ischemia-reperfusion injury (IR) ).

Figure 2. The activity of CD73 / 5'NT on the lungs is inversely correlated with the activity of the disease. Mice CD73 / 5NT - / - and their wild-type littermates (TN),) underwent a false operation or 30 minutes of intestinal ischemia followed by 240 minutes reperfusion (by LPA). In other groups, animals exposed to LPA were previously treated with IFN-�. (A) Activity of CD73 / 5'NT on the lung (mean value ± standard error, mmol of hydrolyzed AMP per milligram of protein per hour) measured in tissue lysates by thin layer chromatography (CCD). (B) Semiquantitative analysis of vascular leakage [(exudation of dextran conjugated with fluorescein-isothiocyanate (FITC)] in the lungs measured in histological sections using image analysis (% of the area of the cut that exhibits fluorescence above a chosen background value arbitrarily, mean values ± standard error) Representative photomicrographs of the indicated groups are also shown Bar, 50 µm. * p <0.05. ** p <0.01.

Figure 3. The activity of CD73 / 5'NT in patients with SIRS suffering from pancreatitis. The data show the activity of CD73 / 5'NT against the degree of severity of pancreatitis: 0 = mild pancreatitis; 1 = severe pancreatitis without organ failure; 2 = severe pancreatitis with organ failure; 3 = sound control.

Figure 4. Activity of CD73 / 5'NT 'versus total stay in the intensive treatment unit (ICU) for patients with all degrees of pancreatitis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and preferred embodiments:

The term "patient" or "individual" refers to a human being or an animal subject.

The term "monitor the development of a disease" means that the worsening of the disease (that is, the worsening of the disease) or the remission of the disease (that is, the recovery of the patient) can be detected by comparing a measured level of biomarker with a control value or one or more previous measurements, performed at different times, of the biomarker level in the same patient. For example, a lower level of the biomarker can be used, compared to the result of a previous measurement or with a control value to indicate the worsening of the disease, while a higher level of the biomarker is used, compared to the result of a previous measure or with a control value to indicate the remission of the disease. However, there are probably certain diseases that affect the biomarker level in the opposite way.

The term "tissue fluid" should be understood to include any liquid that bathes and circles cells. The expression includes, for example, blood plasma, serum, lymph, urine, exudates (pleural, peritoneal) and cerebrospinal fluid.

The term "inflammatory state" is intended to include any harmful and unwanted inflammatory response of an individual's tissue, wherein said inflammatory state may be the result of an acute condition such as tissue trauma, reperfusion injury resulting from myocardial infarction or stroke, organ transplantation or other surgical operation or for a chronic condition including allergic conditions, autoimmune diseases, and inflammatory diseases.

Diseases whose development can be monitored using CD73 protein in tissue fluid are typically selected from the group comprising:

a) tissue trauma

b) reperfusion injury resulting from myocardial infarction or stroke, organ transplantation or any other

surgery,

c) cancer or cancer metastasis, and

d) an inflammatory state.

Typical diseases that lead to a change in the level of CD73 in tissue fluids of patients especially in the serum are: tissue trauma; reperfusion injuries resulting from myocardial infarction or stroke, organ transplantation or other surgical operations, cancer or cancer metastasis; or inflammatory conditions resulting from the aforementioned injuries or reperfusion injuries or chronic conditions including allergic conditions, autoimmune diseases, and inflammatory diseases. Examples of such chronic conditions may be mentioned arthritis, allergic conditions, such as asthma, inflammatory conditions, such as inflammatory bowel disease or an inflammatory skin condition, psoriasis, Parkinson's disease, Alzheimer's disease, autoimmune diseases, type I or type II diabetes, atherosclerosis, multiple sclerosis, Crom disease, or rejection reactions due to transplantation of organs Particularly, inflammatory diseases systemic inflammatory response syndrome (SRIS), acute lung injury (LPA), multiorganic insufficiency (IMO), ischemia reperfusion injury (LIR) and adverse drug reaction (RAM) will lead to alterations of the CD73 protein in tissue tissue.

The term "cytokine" includes any protein or peptide used in organisms as signaling compounds. In particular, this term refers to an interferon or an interleukin, but is not restricted to them. In the event that the cytokine is an interferon, said interferon can be alpha-, beta-, gamma-, omega- or any other interferon and can be any subtype of the aforementioned interferons. Interferons are used in the treatment of the aforementioned diseases. Examples of interleukins can be mentioned IL-4, IL-10, IL-13 and IL-20.

"Statins" form a class of hypolipidemic agents used to lower cholesterol levels in individuals, particularly to reduce the risk of cardiovascular disease. Inflammatory conditions, dementia, cancer, nuclear cataracts and pulmonary hypertension can also respond to statin treatment.

The determination of the CD73 protein in a sample extracted from an individual's tissue fluid can be carried out by immunodetection by quantifying the level of the CD73 protein in said sample by subjecting the sample to a binder or binding agent that recognizes the CD73 protein, and quantify said binder.

Alternatively, detection can be carried out by detecting the activity of the CD73 protein in said sample using thin layer chromatography or by subjecting said sample to the action of a CD73 substrate and monitoring the change of said substrate.

The term "binder" should be understood to include antibodies, which may be monoclonal or polyclonal or modified by genetic engineering; any antibody fragments; aptamers and afficibers, and any other binder capable of binding to an epitope of the CD73 protein. Antibodies to CD73 are well known in the art, see for example http://www.biocompare.com/matrixse/3194/6/67151/CD73.html. The afficuerpos represent a new class of binders and are small and especially stable proteins, developed by Affibody Ab.

The binding assay (agglutination) can be competitive or non-competitive. A preferred assay is a sandwich assay wherein a capture antibody (or other binder class) immobilized on a solid support is subjected to a sample comprising the antigen, which is joined by a first epitope to capture the antibody and add an antibody. labeled (or other class of binder), directed to another epitope of the antigen. The labeled antibody is quantified either directly (homogeneous assay) or after separation of non-immobilized labeled antibodies. The label can be a radioactive isotope, a fluorescent dye, an enzyme or any other detectable label.

For example, for immunodetection purposes, any specific anti-CD73 antibody can be used to capture soluble CD73 from the sample, and then the amount of bound protein can be quantified using a variety of techniques. For example, a sandwich ELISA assay in which an anti-CD73 antibody is immobilized at the bottom of multi-well plates can be employed, the sample is added, and bound CD73 is detected using another anti-CD73 antibody. The anti-CD73 antibody is then detected using any of the multiple techniques suitable for the detection of antibodies, such as labeled second stage antibodies. The specificity for the CD73 of the reaction is controlled by including an irrelevant antibody such as a capture or detection antibody and comparing the signals between these negative controls and the anti-CD73 antibodies.

Determination of CD73 activity

CD73 activity can be measured using thin layer chromatography according to published protocols. CD73 activity can also be measured using an enzymatic assay that measures the conversion of AMP, or other purine mononucleotide that can be used as a CD73 substrate, in the corresponding nucleoside. For example, the assay may be based on the conversion of radioactively or fluorescently labeled substrates. The detection methods may be based on the quantification of the decrease in a substrate concentration or an increase in the product concentration or the release of the phosphate group. The dependence of CD73 on the reaction can be determined by performing the assay in the presence and absence of a known CD73 inhibitor, such as AMPCP.

Known substrates for CD73 are, for example, nucleoside-5'-monophosphates including adenosine-5'monophosphate (AMP), inosine-5'-monophosphate (IMP) and the like.

The invention will be illustrated by the following section of non-restrictive examples.

EXPERIMENTAL SECTION

Materials and methods

Model of acute lung injury (APL) and vascular leakage

CD73 - / - mice were used, which were backcrossed with others of C57BL / 6 origin for 8 generations and wild-type C57BL / 6 (TN) mice. These mice lacked the CD73 protein RNA with enzymatic activity, [3]. The animals were grouped by equality of weight, sex and age. All mice had access to standard food for mice and water until the end of the experiment.

Mice were anesthetized with ketamine hydrochloride (100 mg / kg body weight, i.p.) and xylazine (10 mg / kg body weight, i.p.). During anesthesia the mice breathed spontaneously with normal air. Before surgery the animals received 1 ml of saline solution subcutaneously to compensate for the loss of fluids before the operation. The superior mesenteric artery was dissected by means of a laparotomy in the central line and occluded by a microvascular clamp for 30 minutes. False-operated animals experienced dissection of the superior mesenteric artery without vascular occlusion. The wound was sutured in a layer. The body temperature of the animals was maintained through the ischemia phase with a heating lamp. After ischemia, the microvascular clamp was released, the wound was sutured and the animals subcutaneously received an additional 1 ml of saline. After 235 minutes of reperfusion the mice received dextran conjugated with FlTC (25 mg / kg body weight in 0.2 ml sterile saline solution; molecular weight: 70,000 D, Molecular Probes). After 240 minutes of reperfusion, mice were sacrificed and tissue samples were collected.

The 30-minute ischemia-240 minute reperfusion protocol is an established and reproducible LPA model [9]. The protocol was approved by the Committee on Animal Ethics of the University of Turku (permit 1597/05 to Sirpa Jalkanen).

The effect of IFN-on the activity and permeability of CD73 was studied using pre-and post-treatment protocols. Subgroups of mice were pretreated with mouse IFN-recombinant (6000 IU s.c. once daily for 3 days before ischemia). In the post-treatment group, the animals had a single bolus of IFN- (20,000 IU) intravenously after the ischemic phase at the beginning of the reperfusion period.

All mice were injected i.v. with dextran conjugated with FITC (70 kDa) 5 minutes before euthanasia. Vascular leakage was determined from three color images taken from randomly chosen fields of lungs of cryosected mice using computer image analysis (Image J).

CD73 activity analysis

The activity of ecto-5'-nucleolidase was analyzed by CCD, as previously described [10]. Briefly explained: the test was carried out with the standard enzyme contained in a final volume of 120 µl of RPMI 1640, lung lysate, 5 mmol / L glycerophosphate, and the indicated concentrations of AMP with the [3H] AMP tracer (specific activity , 18.6 Ci / mmol; Amersham, Little Chalfont, United Kingdom). Incubation times were chosen to ensure linearity of the reaction over time, so that the amount of converted AMP did not exceed 7-10% of the initially introduced substrate. Aliquots of the mixture were applied to Alugram SIL G / UV254 CCD sheets (Machercy-Nagel, Düren, Germany) and separation was carried out with isobutanol / isoamyl alcohol / 2-ethoxyethanol / ammonia / H2O (9: 6: 18: 9:15) as solvent. The 3H-labeled AMP and its dephosphorylated nucleoside derivatives were visualized in UV light and quantified using a Wallac-1409 spectrometer. CD73 activity was expressed as nM of hydrolyzed AMP per milligram of protein per hour. The protein concentration in the lysates was determined with the protein analysis kit with bicinconinic acid (abbreviated BCA by the English expression bicinconinic acid) (Pierce, Rockford, IL) according to the manufacturer's instructions.

Statistical Valuations

The non-parametric one-way ANOVA software program (Kruskall-Wallis and Mann-Whitney U tests) was used.

Results

CD73 activity is correlated with disease activity.

Intestinal ischemia-reperfusion (IR) caused marked tissue damage in both the intestine and lungs (Fig. 1). In the lungs of the wild-type mouse falsely operated the activity of CD73 was low (Fig. 2A). Microscopic analysis of FITC-dextran in the lungs showed that there was only a marginal leak in the TN mice undergoing the false operation (Fig. 2B).

When LPA was induced in TN animals, CD73 activity was reduced by 25%. At the same time the vascular leak increased significantly. Changes in vascular permeability are directly correlated with the severity of the disease, since the leakage of intravascular fluids to the pulmonary parenchyma and also to the alveoli is the main cause of deterioration of pulmonary function and impaired gas exchange.

As expected, the activity of CD73 was undetectable or at extremely low levels in mice deficient in CD73 both after the false operation and after the intestinal IR. In CD73-deficient mice, leakage in falsely operated animals was moderately increased when compared to falsely operated wild-type mice. Notably, when LPA was induced, CD73-deficient mice showed approximately 80% more leakage in the lungs than their littermates of TN mice (p = 0.03).

These data show that there is an inverse correlation between CD73 activity and vascular permeability, which is a measure of the severity of the disease.

CD73 activity is correlated with the response to treatment

Previous treatment for 3 days with IFN- (at a dose used clinically in the treatment of multiple sclerosis) led to a 230% increase in the activity of CD73 in lungs of TN mice during APL (p = 0.002, Fig. 2A). More surprisingly, the area of leakage in the TN mice after induction of LPA was reduced by more than 90% after previous treatment with IFN-when compared with untreated littermates (p = 0.0001, Fig. 2B). In fact, there was no difference from the animals only subjected to the false operation. Surprisingly, IFN-also had no protective effects on mice with CD73-deficient LPA. These data show that interferon beta treatment decreases vascular leakage in a surprisingly CD73 dependent manner. In addition, the increase in CD73 activity in wild-type (TN) mice can be used to predict a beneficial outcome in the response to IFN- treatment.

The inventors then analyzed whether IFN-treatment could reverse an established capillary lesion. For this purpose, we treated the mice with IFN-only after the ischemic period at the time of reperfusion. Notably, the single dose of IFN-significantly high improved vascular barrier function during the 4 hours following the reperfusion period. The FITC-dextran leak was reduced by 90 ± 9% in the post-treatment group when compared to the control group (n = 8-13 mice / group, p <0.001). At the same time, the measure of CD73 activity in serum samples increased by more than 30% (from 427 ± 22 in the group with untreated APL to 561 ± 48 in the group with LPA treated with IFN-; p = 0.04, n = 4 / group). Therefore, the induction of CD73 activity correlates positively with a response to treatment. In addition, measuring the activity of CD73 in a blood sample can provide useful information about the ability to respond to IFN-.

References

one.

Hasko, G. and Cronstein, B. N., Adenosine: an endogenous regulator of innate immunity. Trends Immunol 2004. 25; 33-39.

2.

Ohta, A. and Sitkovsky, M., Role of G-protein-coupied adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 2001. 414: 916-920.

3.

Thompson, LF, Elzschig, HK, Ibla, JC, Van De Wiele, CJ, Resta, R., Morote-García, JC and Colgan, SP, Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia . J. Exp. Med. 2004. 200: 13951405.

Four.

Lennon, P. F., Taylor, C. T., Stahl, G. L. and Colgan, S. P., Neutrophil-derived 5'-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A2B receptor activation. J. Exp. Med. 1998. 188: 1433-1443.

5.

Synnestvedt, K., Furuta, GT, Comerford, KM, Louis, N., Karhausen, J., Eltzschig, HK, Hansen, KR, Thompson, LF and Colgan, SP, Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. Clin. Invest. 2002. 110: 993-1002.

6.

Henttinen, T., Jalkanen, S. and Yegutkin, G. G., Adherent Ieukocyles prevent adenosine formation and impair endothelial barrier function by Ecto-5'-nucleotidase / CD73-dependent mechanism. J. Biol. Chem. 2003. 278; 24888-24895.

7.

Eckle, T., Fullbier, L., Wehrmann, M., Koury, J., Mittelbronn, M :, Ibla, J., Rosenberger, P. and Eltzschig, HK, Identification of Ectonucleotidases CD39 and CD73 in lnnate Protection during Acute Lung Injury J. Immunol. 2007. 178: 8127-8137.

8.

Niemela, J., Henttinen, T., Yegutkin, GG, Airas, L., Kujari, AM, Rajala, P. and Jalkanen, S., IFN-alpha induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto -5'-nucleotidase) upregulation. J. lmmunol. 2004. 172; 1646-1653 .:

9.

Ohara, M., Unno, N., Mitsuoka, H ,, Kaneko, H. and Nakamura, S .; Peritoneal lavage with oxygenated perfluorochemical preserves intestinal mucosal barrier function after ischemia-reperfusion and ameliorates lung injury. Crit.Care Med .. 2001. 29: 782-788.

10.

Yegutkin, G. G., Henttinen, T. and Jalkanen, S., Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucecotide kinase activities via fosfotransfer reactions. Faseb J. 2001, 15: 251-260.

eleven.

Airas, L., Niemelä, J., Yegutkin, G. and Jalkanen, S., Mechanism of Action of IFN-f in the treatment of Multiple Sclerosis. Ann N.Y. Acad. Sci. 1110: 641-648 (2007).

12, S. M. Johnson, S. Patel, F. E. Bruckner and D. A. Collins, 5'-Nucleotidase as a marker of both general and local inflammation in rheumatoid arthritis patients. Rheumatology, 1999; 38: 391-396.

Claims (2)

1. A method for monitoring the development of a disease in a patient, wherein said disease is selected from the group comprising: a) tissue trauma 5 b) reperfusion injury as a result of myocardial infarction or stroke, organ transplantation or any another surgical operation, c) cancer or cancer metastasis, and d) an inflammatory state. and where the CD73 extracted from a tissue fluid of said patient is used as a biomarker, wherein the The method is repeated in two or more times and the CD73 level of the sample is compared with that of a previous analysis, and is used to indicate the worsening of the disease or to indicate the remission of the disease, where an increase of the level of CD73 indicates remission of the disease and a decrease in the level of CD73 indicates worsening of the disease. 2. The method according to claim 1, wherein the disease is an inflammatory disease. 3. The method according to claim 2, wherein the disease is systemic inflammatory response syndrome (SRIS), acute lung injury (LPA), multiorganic insufficiency (IMO), ischemiareperfusion injury (LIR) or adverse drug reaction (RAM).